Apparatus and method for immobilizing molecules onto a substrate

ABSTRACT

Apparatus and method for immobilizing molecules, particularly biomolecules such as DNA, RNA, proteins, lipids, carbohydrates, or hormones onto a substrate such as glass or silica; patterns of immobilization can be made resulting in addressable, discrete arrays of molecules on a substrate, having applications in bioelectronics, DNA hybridization assays, drug assays, etc.

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by a grant from the National Science foundation under Grant No.Phy-9408905. The Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to an apparatus and method forimmobilizing a molecule, particularly a biomolecule such as DNA, RNA, ora protein, onto a substrate. Such a substrate has applications inbio-molecular networks, biochip microfabrication, bioelectroniccircuitry, and rational drug design procedures.

BACKGROUND OF THE INVENTION

In many presently used bioassays, a molecule, such as DNA or a protein,is immobilized onto a substrate. For example, Western blots immobilize aprotein onto a substrate, such as PVDF or nitrocellulose membrane, whichis then probed with a ligand to the protein, such as an antibody, todetermine whether the protein is present in a sample. Furthermore,Southern and Northern blots utilize a similar process, wherein DNA andRNA respectively are immobilized onto a nitrocellulose substrate, andprobed with nucleic acid molecules having a complementary sequence tothe desired DNA or RNA. In yet another example, an enzyme-linkedimmunosorbent assay (ELISA) involves immobilizing an antibody onto asubstrate, and then exposing the immobilized antibody to a sample whichis suspected of containing the antigen. If the antigen is present, itwill bind to the immobilized antibody, and that binding can subsequentlybe detected. The methods described above employ primarily hydrophobicinteractions between the molecule and substrate to immobilize themolecule. Hence, successful use of these techniques is dependent uponthe chemical properties of the substrate.

Although these methods are widely used and accepted, they containinherent limitations which can be detrimental to the productivity ofresearchers. For example, immobilizing a molecule onto a substrate is avery time consuming process. Initially, the molecules mustelectrophoresed using SDS-PAGE or agarose gel electrophoresis, whichinvolves pouring the gel (and in the case of SDS-PAGE, exposing theresearcher to toxic bis-acrylamide), installing the gel into a cassette,preparing numerous buffers, preparing the samples for electrophoresis,loading the samples onto the gel, and then electrophoresing the samples.

Immediately after the sample has been electrophoresed and molecules ofthe sample separated by size, the molecules must be transferred to asubstrate, and immobilized onto the substrate. This immobilizationinvolves the preparation of numerous buffers, careful handling of thesubstrate and gel, and carefully contacting the substrate to the gel inthe presence of a buffer to avoid the entrapment of air bubbles betweenthe gel and the substrate. A force, such as an electric current is thenused to transfer molecules from the gel to the substrate.

More recently, efforts have been made to produce chips upon whichmolecules such as proteins or DNA are immobilized in predeterminedarrays. Patterning such molecules on semiconductor substrates, coupledwith specific recognition, are essential for the realization ofbio-molecular networks. Furthermore, addressable arrays of DNA [Fodor,S. P. A., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T., and Solas,D. Science 251:767 (1991); and Southern, E. M. Trends in Genetics 12:110(1996) both of which are hereby incorporated by reference in theirentireties] or proteins immobilized on a substrate can be used toprovide tools for information retrieval, hybridization of DNA andbinding affinity for molecules such as proteins, antibodies, lipids orcarbohydrates in quick and reliable manner.

One method of producing such chips borrows substantially fromphotolithographic microfabrication techniques developed and optimized bythe computer microprocessor industry, which permit the economicproduction of large batches of chips using photographic templates. Morespecifically, current lithographic approaches for immobilizingmolecules, particularly biomolecules onto a substrate, use chemicalmethods to specifically treat substrates with photoresist and then usingphotomasking, a light beam or an electron beam to define the pattern ofimmobilization of the molecule on the substrate. Examples of the use ofthis technique include microlithography on self-assembled monolayers andlipids, microcontact printing, microfluid networks and light directedcombinatorial synthesis [Prime, K. L., and Whitesides, G. M. Science252:1164 (1991); Dulcey, C. S. et al., Science 252:551 (1991); Berggren,K. K., et al., Science 269:1255 (1995); Jackman, R. J.,et al., Science269:664 (1995); Healey, B. G., et al., Science 269:1078 (1995);Delamarche, E., et al., Science 276:779 (1997); Groves, J. T., et al.,Science 275:651 (1997); and Burke, D. T., et al., Genome Research 7:189(1997), all of which are hereby incorporated by reference in theirentireties].

However, all of the above mentioned approaches contain inherentlimitations in that they all depend either on photolithographictechniques or substrate chemistry. For example, a proper control of thesubstrates enables the sequential synthesis of oligonucleotides [Fodor,1991].

Hence, what is needed is an apparatus for immobilizing molecules, suchas biomolecules, onto a substrate which permits patterning of theimmobilization of molecules in adressable arrays, so that the substratesproduced with the apparatus have ready applications in the production ofbio-molecular networks, and to provide tools for information retrieval,hybridization of DNA and binding affinity of ligands for molecules, suchas proteins, DNA, RNA, lipids, or carbohydrates immobilized on thesubstrate.

What is also needed is a method of immobilizing a molecule onto asubstrate reliably and economically, and is not dependent upon thechemistry of the substrate.

The citation of any reference herein should not be construed as anadmission that such reference is available as "Prior Art" to the instantapplication.

SUMMARY OF THE INVENTION

There are provided, in accordance with the present invention, anapparatus and method for immobilizing a molecule onto a substrate thatare simpler and more cost effective than methods cited above, areindependent of substrate chemistry, have biological specificity at themicron scale, and can lead to miniaturization and integration withelectronic components.

Broadly, the present invention extends to an apparatus for immobilizinga molecule onto a substrate, comprising means for directing a beam ofelectromagnetic radiation at a substrate, wherein the substratecomprises a first surface and a second surface and a film of a materialdeposited onto the second surface, wherein the film absorbselectromagnetic radiation and is a poor conductor of heat. Furthermore,the apparatus comprises a chamber for holding a colloidal dispersioncomprising insoluble particles coated with the molecule, such that thecolloidal dispersion is in contact with the film, so that the beam canimpinge the first surface, traverse the substrate, impinge an area ofthe film and ablate and melt the film at the area so that a gas bubbleis formed in the colloidal dispersion above the area. The gas bubble canthen disappear upon displacement of the beam from the area, and theparticles of the colloidal dispersion can adhere to the film at thearea. As a result, the molecule is immobilized onto the substrate.

In a further embodiment, the present invention extends to a method forimmobilizing a molecule onto a substrate, comprising the steps of:

a) providing a substrate having a first surface and a second surface,and a film of a material deposited onto the second surface, wherein thematerial absorbs electromagnetic radiation and is a poor conductor ofheat;

b) contacting the film with a colloidal dispersion comprising insolubleparticles coated with the molecule;

c) directing a beam of electromagnetic radiation towards the substrate,such that the beam impinges the first surface, traverses the substrate,impinges an area of the film and ablates and melts the film in the area,forming a gas bubble in the colloidal dispersion above the area; and

d) displacing the beam so that the gas bubble disappears and particlesadhere to the film at the area, whereby the molecule is immobilized ontothe substrate.

The present invention further extends to an apparatus or method forimmobilizing a molecule onto a substrate, as described above, whereinthe substrate comprises glass or silica. Also, the film deposited ontothe second surface of the substrate can have any thickness. Preferably,the film has a thickness of approximately 50 Å.

The film can be comprised of any material that absorbs electromagneticenergy, is a poor conductor of heat, and has a low melting temperature,i.e. less than 1600 K. Examples of such materials having applications inthe present invention include, but are not limited to gold or aluminum.

Moreover, in an apparatus or method for immobilizing a molecule onto asubstrate, as described above, the colloidal dispersion comprises anaqueous solution, such as an aqueous solution of 50 mM NaCl, 0.02% Tween(polyoxyethylensorbitan, including fatty acid esters thereof), 2 mMNaN₃, pH=7.4. However, numerous other aqueous solutions well known tothose of ordinary skill in the art have applications in the presentinvention. In addition, insoluble particles of the colloidal dispersionmay be made of polystyrene, gold, or glass. The size of the insolubleparticles can range from approximately 2 nm to approximately 100 nm.Preferably, the insoluble particles have a size smaller than the area ofthe film upon which the beam impinges. More particularly, the insolubleparticles comprise polystyrene particles having a size of aboutapproximately 40 nm.

Furthermore, the present invention extends to an apparatus or method forimmobilizing a molecule onto a substrate, as set forth above, whereinthe concentration of insoluble particles in the colloidal dispersion isapproximately 10¹¹ particles/μl.

The present invention further extends to an apparatus or method forimmobilizing a molecule onto a substrate, wherein the beam ofelectromagnetic radiation has a wavelength in the near infrared regionof the electromagnetic spectrum. Examples of applicable wavelengths forthe beam that have applications in the present invention include, butare not limited to 830 nm or 1064 nm. In a preferred embodiment, thebeam is a laser beam comprising a wavelength of 830 nm, an incidentpower of approximately 35 mW, a maximum power of approximately 150 mW,and a circular cross sectional area with a diameter of approximately 1μm.

Moreover, the present invention extends to an apparatus for immobilizinga molecule onto a substrate, as described above, wherein the directingmeans comprises a microscopic objective lens immersed in oil.Preferably, the lens has a magnification of 100X, and a focal point onthe film. In this way, the beam is altered to preferably have a circularcross sectional area with a diameter of approximately 1 μm, andsubstantially all the energy of the beam is delivered to the film.

Similarly, the present invention extends to a method for immobilizing amolecule onto a substrate, as described above, wherein the directingstep comprises propagating the beam through a microscopic objective lensimmersed in oil, wherein the lens has a magnification of 100X, 1.3numerical aperture (NA), and a focal point on the film.

In another embodiment, the present invention extends to an apparatus forimmobilizing a molecule onto a substrate, as described above, whereinthe means for directing a beam of electromagnetic radiation at asubstrate further comprising a means for steering the beam to permitmovement of impingement of the beam on the film. As a result, patternsof immobilization of the molecule onto the substrate can be made. Anexample of a steering means of the present invention involves the use oftwo telescopic lenses each with a focal length of 100 mm. These lensesare positioned such that the beam passes through both lenses, and can bemoved in three dimensions (X,Y, and Z). As a result, the area of thefilm upon which the beam impinges can be controllably changed,permitting the creation of patterns of immobilization of the moleculeonto the substrate. The velocity of movement of the impingement of thebeam on the film can vary, and is dependent upon the application and theamount of molecule one wishes to immobilize in a particular area of thefilm. More particularly, the slower the beam is steered, the longer itis permitted to impinge on a particular area of the film, and thegreater the amount of molecule immobilized to that particular area offilm. Preferably, the velocity of the movement of impingement of thebeam with the film ranges from approximately 5 μm/second toapproximately 50 μm/second.

Naturally, the present invention extends to a method for immobilizing amolecule onto a substrate, as described above, wherein the directingstep further comprises steering the beam to permit movement ofimpingement of the beam on the film. As a result, patterns ofimmobilization of the molecule onto the substrate can be made. Anexample of steering the beam pursuant to the present invention involvespropagating the beam through two telescopic lenses each with a focallength of 100 mm. These lenses are positioned such that the beam passesthrough both lenses, and the impingement of the beam onto the film canbe moved in X and Y directions. As a result, the area of the film uponwhich the beam impinges can be controllably changed, permitting thecreation of patterns of immobilization of the molecule on the substrate.Furthermore, the velocity of movement of the impingement of the beam onthe film can vary, and is dependent upon the application and the amountof molecule one wishes to immobilize in a particular area of the film.More particularly, the slower the beam is steered, the longer it ispermitted to impinge on a particular area of the film, and the greaterthe amount of molecule immobilized to that particular area of film.Preferably, the velocity of the movement of impingement of the beam withthe film ranges from approximately 5 μm/second to approximately 50μm/second.

The present invention further extends to an apparatus for immobilizing amolecule onto a substrate, as set forth herein, further comprising ameans of generating the beam of electromagnetic energy. As explainedinfra, the beam can have any wavelength, provided the film absorbs theenergy of the beam at the area of the film upon which the beam impinges,has a low melting temperature, i.e., less than 1600 K, and is a poorconductor of heat, i.e., heat generated in the area of impingement ofthe beam onto the film remains localized at the area of impingement. Onexample of a means for generating a beam which has applications in thepresent invention is a laser. For example, should a laser beam having awavelength of 1064 nm be desired, an Nd YAG laser, which is commerciallyavailable, can be used in the present invention. In a preferredembodiment, the laser is a laser diode producing a beam having awavelength of 830 nm.

Likewise, the present invention extends to a method for immobilizing amolecule onto a substrate, further comprising the step of generating thebeam of electromagnetic energy prior to the step of directing the beamto the substrate. Pursuant to the present invention, the beam can haveany wavelength, provided the film absorbs the energy of the beam at thearea of the film upon which the beam impinges, and has a low meltingpoint, and is a poor conductor of heat. As a result, the heat generatedin the film remains localized at the area of impingement. Generatinglaser beams with various wavelengths is well known to one of ordinaryskill in the art.

Furthermore, the present invention, as described herein, can be used toimmobilize numerous types of molecules onto a substrate in adressablepatterns. For example, biomolecules such as a nucleic acid molecule orfragments thereof, an isolated protein or fragments thereof, a lipid, ora carbohydrate, to name only a few, can be immobilized onto a substratewith the apparatus of the present invention. More specifically examplesof nucleic acid molecules or fragments thereof which can be immobilizedinclude, but are not limited to, DNA, RNA, or a combination of DNA andRNA. Furthermore, such nucleic acid molecules can be double stranded orsingle stranded. In addition, numerous examples of proteins, includingantibodies, receptor proteins, DNA binding proteins, proteins having aprotein binding domain, cytokines, lymphokines, or hormones, to nameonly a few, can be immobilized onto a substrate using the presentinvention.

In another embodiment, the present invention extends to an apparatus andmethod for immobilizing a molecule onto a substrate, wherein anindividual insoluble particle of a colloidal dispersion, and coated withthe molecule, is grafted onto the substrate. As a result, a particularpattern of immobilization of the molecule, or multiple molecules, can bemade.

The present invention further extends to an apparatus for immobilizing amolecule onto a substrate, comprising:

a) a chamber for holding a colloidal dispersion of insoluble particlescoated with the molecule;

b) means for supporting the substrate in the chamber; and

c) means for selecting a particle of the colloidal dispersion andgrafting the particle onto the substrate, such that the molecule isimmobilized onto the substrate.

In addition, the present invention extends to a method of immobilizing amolecule onto a substrate, comprising the steps of:

a) providing a chamber for holding a colloidal dispersion of insolubleparticles coated with the molecule;

b) supporting the substrate in the chamber; and

c) selecting a particle of the colloidal dispersion;

d) grafting the particle onto the substrate, such that the molecule isimmobilized onto the substrate.

Hence, with such an apparatus and method of the present invention, asingle particle coated with a particular molecule can be grafted to asubstrate. As a result, patterns of immobilization of a molecule ormultiple molecules can be created on the substrate.

Furthermore, the present invention extends to an apparatus or method forimmobilizing a molecule onto a substrate in which a particle of acolloidal dispersion is grafted onto the substrate, wherein thesubstrate comprises a cantilever, and the particle is grafted to thecantilever. Examples of substrates having applications in the presentinvention include, but are not limited to silicon and silicon nitride.

In addition, a film comprising a material that may be deposited onto thesubstrate, wherein the material absorbs electromagnetic radiation, is apoor conductor of heat, and has a low melting temperature, i.e., lessthan 1600 K. Numerous materials have applications in the presentinvention as a film, including gold or aluminum, to name just a few. Ina preferred embodiment, the film has a thickness of approximately 50 Å.

The present invention further extends to an apparatus or method forimmobilizing a molecule onto a substrate in which a particle of acolloidal dispersion is grafted onto a substrate, as set forth above,wherein the colloidal dispersion comprises an aqueous solution. Numerousaqueous solutions can be used, particularly those that enhance theviability and activity of biomolecules. In a preferred embodiment, theaqueous solution comprises 0.1 M PBS. However, other solutions readilyapparant to one of ordinary skill in the art also have applications inthe present invention.

In addition, particles of a colloidal dispersion of the presentinvention coated with a molecule can be comprised of numerous materials,such as polystyrene, glass or gold, to name only a few. In a preferredembodiment, the particles are comprised of polystyrene and have a sizeof approximately 3 μm.

The present invention extends to an apparatus for immobilizing amolecule onto a substrate, comprising:

a) a chamber for holding a colloidal dispersion of insoluble particlescoated with the molecule;

b) means for supporting the substrate in the chamber; and

c) means for selecting a particle of the colloidal dispersion andgrafting the particle onto the substrate, such that the molecule isimmobilized onto the substrate, wherein the selecting and grafting meanscomprises an optical tweezers. An example of an optical tweezer havingan application in the present invention is described in Ashkin, A.,Dziedzic, J. M., Bjorkholm, J. E., and Chu, S., Observation of asinglebeam gradient force optical trap for dielectric beads. Opt.Lett.11:288-290 (1986), which is hereby incorporated by reference in itsentirety. Broadly, an optical tweezer comprises a laser beam having anda microscopic objective lens, such that the beam passes through thelens, and enters the chamber. In a preferred embodiment of theinvention, the laser beam of the tweezer comprises a wavelength in thenear infrared region of the electromagnetic spectrum, such as 830 nm,and is produced by a near infrared laser diode. Furthermore, the lens ofan optical tweezer having applications in the present invention as amicroscope objective lens with properties of 100 X, and 1.3 numericalaperture (NA).

Furthermore, the present invention extends to a method for immobilizinga molecule onto a substrate, comprising the steps of:

a) providing a chamber for holding a colloidal dispersion of insolubleparticles coated with the molecule;

b) supporting the substrate in the chamber;

c) selecting a particle of the colloidal dispersion; and

d) grafting the particle onto the substrate, such that the molecule isimmobilized onto the substrate, in which the selecting and graftingsteps are performed with an optical tweezer. An optical tweezer havingapplications in the invention has been described above.

Moreover, in an embodiment an apparatus of the present invention, thechamber for holding a colloidal dispersion of insoluble particles coatedwith the molecule to be immobilized is formed by a #1 coverglass with apolyvinyline o-ring glued onto the coverglass with paraffin, and thesupporting means for supporting the substrate in the chamber comprisesan XYZ stage adjacent to the chamber. This stage permits the movement ofthe substrate towards the selected particle in the optical tweezer. As aresult, the beam of the optical tweezer impinges an area of thesubstrate, and particularly the cantilever of the substrate. The area ofimpingement is then heated, so that when the beam is displaced, theparticle can adhere to the area, and the molecule is immobilized on thesubstrate.

Naturally, the present invention extends to a method for immobilizing amolecule onto a substrate, as set forth herein, wherein the step ofproviding chamber comprises providing a #1 coverglass with apolyvinyline o-ring glued onto the coverglass with paraffin, Inaddition, the supporting step of a method of the present inventioninvolves providing an XYZ stage adjacent to the chamber, which permitsthe movement of the substrate towards the selected particle in theoptical tweezer.

Accordingly, it is a principal object of the present invention toprovide an apparatus and method for immobilizing a molecule, such as abiomolecule onto a substrate, which permits the formation of discrete,addressable patterns of immobilization of molecules on a substrate.These substrates can then be used in numerous applications, including inbio-molecular networks, bio-electronic circuitry, and in hybridizationassays, protein assays, and drug design.

Another object of the present invention is to provide a simple,reliable, and inexpensive methods of immobilizing desired molecules ontoa substrate, which requires very small quantities of molecules.

It is yet another object of the present invention to immobilizemolecules, particularly biomolecules such as DNA, RNA, proteins, orcarbohydrates, to name only a few, without modulating the activity ofsuch molecules.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematical view of an apparatus and method of the presentinvention. A laser beam is focused onto a gold-coated glass substrate.Laser absorption results in local melting and ablation of the gold film.Using a laser beam steering, based on a telescopic lens system, thefocused spot is moved in the x-y plane to obtain any desired pattern.Inset a. Top: a line pattern RU (Rockefeller University). Line widthless than 1 μm. The pattern is visualized using epi-illumination and aCCD camera. Bottom: array of spots with spacing of about 2 μm. In bothcases the negative of the images are presented for better contrast.

FIG. 2. Schematical view of probable particle motion in the colloidaldispersion. Although not intending to be bound by any particular theory,Applicants believe that due to convective flow, particles flow in thecolloidal dispersion as set forth herein.

FIG. 2B. Schematical view of the particle aggregation on the laserablated substrate.

FIG. 2C. Fluorescence visualization of the adsorbed pattern. Width ofthe line pattern depends on laser power, spot size, rate of inscriptionand the concentration of particles in suspension. On top a continuousline and a discontinuous one (laser blocked for a short time) are shown(5 μm/sec thick line, 10 μm/sec middle line, 20 μm/sec thin line, 35 mWpower).

FIG. 3. Schematical view of the steps in biospecific lithography andrecognition.

FIG. 3A is a schematical view of avidin coated particles suspended in acolloidal dispersion.

FIG. 3B is a schematical view of avidin coated particles adhering to thefilm of the substrate, such that avidin is immobilized onto thesubstrate in a pattern.

FIG. 3C is a schematical view of recognition by a second set of biotincoated fluorescent particles.

FIG. 3D is a fluorescence image after specific recognition of thebiotin-avidin complex.

FIG. 3E shows fluorescence images after specific recognition of thebiotin-avidin complex with varying line widths obtained by changing thefocal height and therefore the size of the area of impingement of thebeam on the film. (shown from top to bottom 1 μm, 3 μm, 5 μm, 7 μm, 60mW power).

FIG. 4. Schematical view of the DNA hybridization experiment.

FIG. 4A is a schematical view of alternating patterns of moleculesimmobilized on a substrate, wherein the patterns are generated by twodifferent single stranded oligomers attached to 40 nm particles.

FIG. 4B is a schematical view of specific recognition in the detectionstep;

FIG. 4C is a fluorescence image detection of the immobilized DNAsequences by complementary single stranded DNA hybridizationfluorescently labeled (succinimidyl ester of carboxyfluorrescein (FAM)for A and Rhodamine for B). Grayscale images are acquired with anintensifier. They arc artificially colored to represent the twodifferent fluorophores and then added.

FIG. 5

FIG. 5A is a schematical view of the second embodiment of the presentinvention (not to scale). A silicon cantilever is used as asemiconductor substrate.

FIG. 5B is a schematical view (not to scale) of grafting a particle to acantilever: an optical tweezer (70 mW incident tweezer laser power, 830nm) is used to trap a 3 μm particle coated with numerous copies of asingle stranded DNA molecule with a known DNA sequence. The particle isthen moved onto the plane of the silicon cantilever.

FIG. 5C depicts a trapped particle, visualized in bright-filed imaging,brought in contact with the cantilever edge. To graft, the tweezer iskept on for less than a second. Bar=5 μm.

FIG. 6

FIG. 6A is a schematical view and micrograph of a particle, covered withsingle stranded DNA oligonucleotides, grafted to the cantilever. Thecantilever tips are shown with particles, using bright field imaging.

FIG. 6B is a schematical view of the hybridization of a oligonucleotidehaving a DNA sequence complementary to the DNA sequence of theoligonucleotide coating the particle, and a fluorescence detection ofthe grafted sequences by complementary DNA hybridization. The greyscaleimages are acquired with an image intensifier and colored to representtwo different fluorophores. The complementary sequence to theoligonucleotide having a DNA sequence of SEQ ID NO:1 is tagged withfluorophore FAM at the 3' end, and the nucleic acid moleculecomplementary to the oligonucleotide having a DNA sequence of SEQ IDNO:2 is tagged with rhodamine at the 3' end. Bar=5 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon Applicants' discovery thatunexpectedly, a substrate in contact with a colloidal dispersioncomprising insoluble particles coated with a molecule can be impingedwith laser beam to form patterns of melting and ablation on thesubstrate to which the insoluble particles can adhere without effectingthe activity of the molecule, particularly biomolecules. As a result,the molecule is immobilized onto the surface of the substrate, andavailable for further use in numerous assays, and bio-electronicapplications.

The present invention is further based upon Applicants' discovery thatunexpectedly, individual particles coated with a molecule can beattached to a substrate at a specific predetermined location.

Hence, broadly the present invention extends to an apparatus forimmobilizing a molecule onto a substrate, comprising means for directinga beam of electromagnetic radiation at a substrate, wherein thesubstrate comprises a first surface and a second surface and a film of amaterial deposited onto the second surface, wherein the film absorbselectromagnetic radiation and is a poor conductor of heat. Furthermore,the apparatus comprises a chamber for holding a colloidal dispersioncomprising insoluble particles coated with the molecule, such that thecolloidal dispersion is in contact with the film, so that the beam canimpinge the first surface, traverse the substrate, impinge an area ofthe film and ablate and melt the film at the area so that a gas bubbleis formed in the colloidal dispersion above the area. The gas bubble canthen disappear upon displacement of the beam from the area, and theparticles of the colloidal dispersion can adhere to the film at thearea. As a result, the molecule is immobilized onto the substrate.

The present invention further extends to a method for immobilizing amolecule onto a substrate, comprising the steps of:

a) providing a substrate having a first surface and a second surface,and a film of a material deposited onto the second surface, wherein thematerial absorbs electromagnetic radiation and is a poor conductor ofheat;

b) contacting the film with a colloidal dispersion comprising insolubleparticles coated with the molecule;

c) directing a beam of electromagnetic radiation towards the substrate,such that the beam impinges the first surface, traverses the substrate,impinges an area of the film and ablates and melts the film in the area,forming a gas bubble in the colloidal dispersion above the area; and

d) displacing the beam so that the gas bubble disappears and particlesadhere to the film at the area, whereby the molecule is immobilized ontothe substrate.

For purposes of the present invention, a material which is a "poorconductor of heat" is a material which, upon impingement with a laserbeam, absorbs the energy of the beam at the area of impingement, andheated generated at the area is not substantially conducted awaytherefrom to other parts of the material. As a result, absorbtion of theenergy of the beam results in local melting and ablation of the film atthe area of impingement.

Furthermore, for purposes of the present invention, a material having a"low melting" temperature refers to a material having a melting pointless than or equal to 1600 K. Examples of such materials include, butare not limited to, gold or aluminum.

Referring to FIG. 1, substrate (1) has a first surface (2) and a secondsurface (3). Examples of materials which can serve as a substrateinclude, but are not limited to glass or silica. In a preferredembodiment, the substrate is comprised of silica having a width ofapproximately 100 μm. In addition, film (4) is deposited onto surface(3) of substrate (1). It is preferred that film (4) be a material thatabsorbs electromagnetic energy, particularly energy in the near infraredregion of the electromagnetic spectrum, is a poor conductor of heat, andhas a low temperature melting point, i.e. less than 1600 K. Examples ofmaterials having applications as film (4) in the present inventioninclude gold or aluminum to name only a few. In a preferred embodiment,film (4) is gold having a thickness of approximately 50 Å. Methods ofdepositing film (4) onto surface (3) of substrate (1) are readily knownto the skilled artisan, and include vapor deposition, plasma enhancedvapor deposition, chemical deposition, or sputtering, to name only afew.

Referring again to FIG. 1, a chamber (5) is provided for holding acolloidal dispersion (6) in contact with film (4) deposited onto surface(3) of substrate (1). Preferably, colloidal dispersion (6) comprisesinsoluble particles coated with the molecule to be immobilized onto thesubstrate, and an aqueous solution. Numerous particles have applicationsin the present invention, and are readily apparent to the skilledartisan. For example, the particles may be comprised of a material suchas polystyrene, gold, or glass to name only a few. Furthermore, numeroustypes of molecules, such as biomolecules can be coated onto insolubleparticles. Examples of biomolecules which can be coated onto insolubleparticles in colloidal dispersion (6) include, but are not limited tosingle or double stranded nucleic acid molecules, such as DNA, RNA, or acombination of DNA and RNA, proteins, such as antibodies, receptors, DNAbinding proteins, cytokines or lymphokines, or proteins having a domainwhich binds to other proteins, hormones, carbohydrates, and lipids.Since methods of coating particles with a molecule, particularly abiomolecule such as DNA, are readily known to one of ordinary skill inthe art, they will not be explained in detail here.

The size of the insoluble particles may vary, and is dependent upon theparticular application. For example, their size may range fromapproximately 2 nm to approximately 100 nm. It is preferred theinsoluble particles of colloidal dispersion (6) have a sizes less thanthat of the area of film (4) upon which laser beam (11) impinges. Thisimpingement will be explained below. In a specific embodiment of thepresent invention, the insoluble particles are comprised of polystyreneand have a size of approximately 40 nm. Furthermore, the concentrationof coated insoluble particles in colloidal dispersion (6) can varydepending upon the application. In an embodiment of the invention, theconcentration of particles in colloidal dispersion (6) is approximately10¹¹ particles/ μl of dispersion.

Moreover, numerous aqueous solutions have applications in colloidaldispersion (6) held in chamber (5). If the molecule to be immobilized isa biomolecule, such as DNA, RNA, or a protein, to name only a few, it ispreferred the aqueous solution be comprised of compositions thatpreserve the activity of the molecule, such as phosphate buffered saline(PBS). An example aqueous solution of colloidal dispersion (6) held inchamber (4) comprises 50 mM PBS, 50 mM NaCl, 0.02% (v/v) Tween, 2 mMNaN₃, pH 7.4. Another example having applications in the presentinvention is 0.1 M PBS (phosphate buffered saline). Other solutions thatmaintain the viability and activity of biomolecules readily apparent tothe skilled artisan also have applications in the present invention.

In addition, as explained above, an apparatus of the present inventionprovides a means for directing a beam (11) of electromagnetic radiationtowards substrate (1), such that the beam impinges surface (2) ofsubstrate (1), traverses substrate (1), impinges an area of film (4) andablates and melts film (4) in the area. In an embodiment of the presentinvention, beam (11) is a laser beam having a wavelength in the nearinfrared region of the electromagnetic spectrum. Examples of wavelengthsfor beam (11) that have applications in the present invention include,but are not limited to 830 nm or 1064 nm. In a preferred embodiment ofthe present invention, beam (11) is a laser beam comprising a wavelengthof 830 nm, an incident power of approximately 35 mW, and maximum powerof approximately 150 mW. A means for directing beam (11) of the presentinvention includes microscopic objective lens (7). In order to increasethe efficiency of the apparatus of the invention, lens (7) should have afocal point on film (4) so that substantially all the energy of beam(11) is delivered to the area of impingement on film (4). Furthermore,lens (7) may be immersed and oil, have a magnification of 100X, a 1.3N.A., and a focal point on film (4). Optionally, a means for directingbeam (11) towards substrate (1) may include mirror (8), should a skilledartisan desired to have beam (11) enter the apparatus of the presentinvention from an angle relative to the focal plane of lens (7).

Again referring to FIG. 1, an apparatus of the present invention mayfurther include a beam steering means (9) which permits the movement ofimpingement of beam (11) on film (4). As a result, the skilled artisancan control the formation of patterns of immobilization of a molecule ormolecules onto substrate (1). In an embodiment of the present invention,steering means (9) comprises two telescopic lenses, each with a focallength of 100 mm (lens not shown). The lenses are positioned such thatbeam (11) propagates through them prior to propagating through lens (7)and impinging an area of film (4). As a result, beam (11) can becontrolled to impinge more than one area of film (4), permitting thecreation of patterns of immobilization of a molecule onto substrate (1).

Moreover, the velocity at which beam (11) is moved, and hence, thevelocity of the change of area of film (4) impinged by beam (11) canvary, depending upon the particular application. The lower the velocityof the movement of beam (11), the longer it is permitted to impinge aparticular area of film (4), and the greater the amount of moleculeimmobilized on the particular area of film (4). The range of velocitiesof movement of beam (11) are approximately 5 μm/second to approximately50 μm/second.

Furthermore, as set forth in FIG. 1, an apparatus of the presentinvention may optionally comprise a means (10) for generating beam (11),such as a laser. As explained above, it is preferred beam (11) have awavelength in the near infrared region of the electromagnetic spectrum.Hence, means (10) having applications in the present invention includean Nd YAG laser diode, which emits a beam having a wavelength of 1064nm, or a laser diode producing a beam having a wavelength of 830 nm.Preferably, means (10) is a laser diode which generates a beam having awavelength of 830 nm.

In addition, as explained above, the present invention extends to amethod for immobilizing a molecule onto a substrate, comprising thesteps of:

a) providing a substrate having a first surface and a second surface,and a film of a material deposited onto the second surface, wherein thematerial absorbs electromagnetic radiation and is a poor conductor ofheat;

b) contacting the film with a colloidal dispersion comprising insolubleparticles coated with the molecule;

c) directing a beam of electromagnetic radiation towards the substrate,such that the beam impinges the first surface, traverses the substrate,impinges an area of the film and ablates and melts the film in the area,forming a gas bubble in the colloidal dispersion above the area; and

d) displacing the beam so that the gas bubble disappears and particlesadhere to the film at the area, whereby the molecule is immobilized ontothe substrate.

Hence, pursuant to the method of the present invention, and referring toFIG. 1, substrate (1) having a first surface (2) and a second surface(3) upon which a film (4) is deposited is provided, a colloidaldispersion (6), having properties as described above, is provided andplaced in contact with film (4) of substrate (1). Optionally, chamber(5) for holding colloidal dispersion (6) in contact with film (4) isprovided. As described above, colloidal dispersion (6) comprisesinsoluble particles coated with the molecule to be immobilized onto thesubstrate, and an aqueous solution. A beam of electromagnetic radiation(11) is then directed at substrate (1) such that beam (11) impingessurface (2) of substrate (1), traverses substrate (1), and impinges anarea of film (4). As a result the area of impingement of film (4) isablated and melted. Furthermore, a bubble forms in colloidal dispersion(6) above the area. After beam (11) is displaced or moved such that itno longer impinges the area of film (4), the bubble disappears andparticles of colloidal dispersion (6) adhere to the area of film (4). Asa result, the molecule is immobilized to substrate (1). Additionaldetails regarding the method of the invention are described below inExample I.

FIG. 2(a) is a schematical view of chamber (5) of the present invention.In particular, beam (11) (not shown) impinges an area of film (4) ofsubstrate (1), such that film (4) is in contact with colloidaldispersion (6). As a result, the area of film (4) impinged by beam (11)(not shown) is ablated and melted, and a bubble forms in colloidaldispersion (6) which prevents insoluble particles (12) coated with themolecule to be immobilized from interacting with film (4). However,after beam (4) is moved or displaced, the bubble above the area of film(4) disappears, and particles (12) can adhere to the area of film (4).As a result, the molecule is immobilized onto substrate (1).

FIG. 2(B) is a schematical view of the adherence of particles (12) ontosubstrate (1). The area impingement of beam (11) (not shown) onto film(4) is schematically indicated with broken lines. After beam (11) (notshown) is displaced or moved, the bubble formed in colloidal dispersion(6) above the area disappears, and particles (12) can adhere to the areaof impingement, as schematically shown in FIG. 2(B).

In another embodiment, the present invention extends to an apparatus forimmobilizing a molecule onto a substrate, comprising:

a) a chamber for holding a colloidal dispersion of insoluble particlescoated with the molecule;

b) means for supporting the substrate in the chamber; and

c) means for selecting a particle of the colloidal dispersion andgrafting the particle onto the substrate, such that the molecule isimmobilized onto the substrate, wherein the selecting and grafting meanscomprises an optical tweezer.

An example of an optical tweezers having an application in the presentinvention is described in Ashkin, a., Dziedzic, J. M., Bjorkholm, J. E.,and Chu, S., Observation of a singlebeam gradient force optical trap fordielectric beads. Opt.Lett. 11:288-290 (1986), which is herebyincorporated by reference in its entirety. Pursuant to FIG. 5, theoptical tweezer comprises beam of electromagnetic radiation (22) andlens (18), such that the beam (22) propagates through the lens (18) andenters chamber (13). In an embodiment of the invention, beam (22) has awavelength in the near infrared region of the electromagnetic spectrum,such as 830 nm, and is produced by near infrared laser diode (19).Furthermore, lens (18) is a microscope objective lens with properties of100 X, and 1.3 N.A.

Again referring to FIG. 5, chamber (13) for holding colloidal dispersion(14) of insoluble particles coated with the molecule to be immobilizedonto substrate (15) is formed by a #1 coverglass with a polyvinylineo-ring glued onto the coverglass with paraffin, and the supporting meansfor supporting substrate (15) in chamber (13) comprises an XYZ stage(17). Stage (17) permits the movement of substrate (15) in chamber (13)towards the selected particle in the optical tweezer. As a result, beam(22) impinges an area of substrate (15), and particularly a cantilever(16) of substrate (15). The area of impingement is then heated, so thatwhen beam (22) is displaced, the optical tweezer can move the selectedparticle towards the area of impingement of substrate (15), permittingthe particle to adhere to the area. As a result, the molecule isimmobilized onto substrate (15), and particularly cantilever (16) ofsubstrate (15).

Optionally, colloidal dispersion (14) can be stored in multiwellparticle chamber (21), which is connected to sample chamber (13) and influid registry therewith via flow cell (24). Also optionally, the meansfor selecting and grafting the particle onto the substrate may includemirror (20), which permits beam (22) to be produced and enter theapparatus of the present invention at an angle to the focal plane ofmicroscope objective (18). Furthermore, the present invention mayoptionally include a fluoresce excitation source (23) which follows thesame path of beam (22) of the optical tweezer, and excites ligandslabeled with a fluorescent chemical which bind to the molecule coatinginsoluble particle of colloidal dispersion (not shown) that has beengrafted to substrate (15) using an apparatus and method of the presentinvention. Hence, the presence of the immobilized molecule on substrate(15) can be detected.

The present invention may be better understood by reference to thefollowing non-limiting examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE I A new method for immobilizing and patterning bio-molecules ona substrate, using localized laser absorption, and sub-micronlithography

Direct beam bio-molecular patterning on gold-coated glass substrates,using diffraction limited near infrared laser spot, is used for DNArecognition and specific ligand-receptor interactions. Using amicroscope objective lens, a near infrared laser beam is focused onto aglass slide coated with 50-Angstrom gold film. Localized laserabsorption results in partial melting and ablation of gold at the areaof impingement of the beam onto the film. Spatially moving the laserspot, a stable etched gold pattern is obtained at submicron resolution.During the process, sub-micron particles in a colloidal dispersion incontact with the film aggregate along the melted gold film. Thesub-micron particles are themselves coated with specific biomolecules,like the protein avidin or single stranded DNA oligomers, for specificbiomolecular recognition. Hence, the present invention can be used tocreate patterns of immobilization of biomolecules on a substrate. Thephysical essentials of the technique are straightforward and simple torealize. A near infrared laser, focused onto a diffraction-limited areaof the film to locally heat a thin gold film deposited on the surface ofa substrate. Ablation of the gold at the area of the film upon which thebeam impinges leaves a permanent trace as the laser spot is translated.Concurrently, a convective flow is induced in the aqueous solution ofthe colloidal dispersion. Insoluble particles coated with the moleculeto be immobilized onto the substrate in the colloidal dispersion areentrained by the flow and locally attracted by the laser, acting as anoptical trap. The particles are protected from overheating by a minutegas bubble, which forms above the area of the film impinged by the laserbeam. As the beam is displaced or moved, the bubble shrinks and theparticles stick to the ablated area of the film. Hence, the molecule isimmobilized onto the film.

If the particles of the colloidal dispersion are conjugated withbiomolecules on their surface, a number of interesting possibilitiespresent themselves. In particular, the present invention can be used toproduce DNA oligonucleotides micro arrays for cDNA libraries. Inaddition, other biomolecules such as proteins (antibodies, cytokines,lymphokines, receptors, DNA binding proteins, proteins having domainswhich bind other proteins), hormones, lipids, or carbohydrates caneasily be immobilized onto a substrate with the present invention.Different geometries, lines, points, arrays can be generated at will aswell as writing. A wide range of particle size can be used, from 10 nmto a few μm, giving great versatility. The main attraction of thepresent invention is the easiness of its implementation forimmobilization and patterning of biomolecules on a solid substrate. Tworealizations are set forth herein. In the first one, a biotin-avidincomplex is used as an example of receptor-ligand specificity. The seconddemonstrates the applicability to DNA recognition through hybridization.

FIG. 1 presents the physical components of an apparatus of the presentinvention. Glass substrates used in the experiment are prepared bysputtering thin gold films on the order of 50 Å. Numerous methods ofdepositing a film onto a substrate are well known to the skilledartisan. The method used herein to deposit the film of gold onto thesubstrate (#1 cleaned glass coverslips, 22 mm²) involved standardsputtering (BAL-TEC MED 020) in argon atmosphere, with the target beingkept at room temperature. Rate of depositions 5 Å/sec. The thickness, 50Å, is measured in vitu using a quartz monitor.

A near infrared laser beam (maximum power 150 mW, 830 nm) is focusedonto a diffraction limited spot on the gold film using a microscopeobjective lens (100X, 1.3 NA, oil immersion), which also serves forvisualization purposes More specifically, a laser beam produced from anear infrared laser diode (SDL Inc.) is collimated to a circular beam of1 μm diameter. Two telescopic lenses (100 mm focal length) are used forbeam steering. To generate patterns smaller than 30 μm, beam steering isused. For larger patterns, the sample stage, on which the coverslip ismounted (not shown in FIG. 1), is translated.

The use of an aqueous solution in the present invention can be avoided,but it has been found that aqueous solutions help to avoid excessheating and enhances the contrast. It is also essential for the nextstep of particle decoration, or adherence to the substrate.

Thin granular films of gold absorb infrared radiation well, but conductheat poorly. Improved thermal isolation is caused by the formation of asmall gas bubble when the gold film is heated. The exposure time to thelaser beam at a given spot is critical, since continuous heating formore than a few seconds results in the formation of large gas bubbles.Exposure times of about 0.2 seconds on a particular area of the film(for 35 mW incident power) ensure that the size of the bubble is on theorder of the beam spot size (i.e., the size of the area of the film uponwhich the beam impinges).

The inset to FIG. 1 shows applications of the present invention. Drawingcontinuous lines produces writing with submicron thickness. An array ofmicron scale dots with micron scale separation is also shown. In anembodiment of the present invention, manual beam steering was used tomove the area of impingement of the beam upon the film. However,automated XYZ translation and beam control can easily be implemented.The inscribed line width can vary from 500 nm to 10 μm and depends onthe size of the area of the film upon which the beam impinges.

The introduction of the insoluble particles coated with the molecule tobe immobilized, in the colloidal dispersion, leads to a number ofeffects, as described in FIG. 2. Local heating of the gold film inducesa convective flow (FIG. 2A), entraining the particles of the colloidaldispersion toward the hot spot, where they are further attracted by thelaser tweezing power. There, a gas bubble forms over the area ofimpingement and acts as a thermal shield so that the particles are neverin contact with the very hot gold area. This reduces the damage to theeventual functionality of the biomolecules absorbed on the particles'surface. Finally, as the beam is displaced, the microbubble disappearsand the particles wet the still warm gold surface, which is then below100° C., and stick to it permanently. In all of this process the flowmaintains the local concentration of particles, and thus no localdepletion is caused by the adhesion. Approximately 6×10⁵ particles arerequired to decorate a line 10 μm wide and 100 μm long. As 10¹¹particles/μl of colloidal dispersion (volume 10 μl) are used,concentration depletion at the etching front during lithography isnegligible. During laser inscription a typical mean flow velocity at thefocal plane is 20 μm/sec (micron-sized particles are used here in orderto visualize).

40 nm size fluorescent particles (10 μl volume and 10¹¹ particles/μl)made of polystyrene were used to decorate the ablated and melted area ofthe film. Numerous sizes of particles have been examined, and it hasbeen determined that optimum results are obtained when the particles aresmaller than the diffraction limited area of the film upon which thelaser beam impinges. The control parameters of the lithographic processset forth herein are: laser power, spot size, particle size andconcentration, rate of inscription. As a line was drawn by moving thesample stage (or beam steering), small z shift of the objective was notautomatically corrected. As a result, there existed a slightinhomogeneity of particle deposition. To pattern biomolecules, a nearinfrared laser is suitable. At this wavelength there is minimal damageto the biomolecules. Furthermore, any substrate may work as long asthere is localized heating and a colloidal dispersion.

For inscription the laser scans at velocities ranging from 5 to 50μm/sec. At the end of the inscription the sample cell is washed toremove free particles (FIG. 2B). As seen in FIG. 2C, the insolubleparticles of the colloidal dispersion decorate the pattern, which can bevisualized by fluorescence techniques, and their density depends on thevelocity of the laser scan. Using high-resolution images, it has beenfound that the decorated lines are composed of micro-domains, with amean diameter on the order of the laser spot.

The first example of an apparatus and method of this embodiment of thepresent invention utilizes 40 nm avidin coated (non-fluorescent)spheres. The presence of a layer of proteins does not alter the wettingand sticking properties of the particles (11). After drawing the patternon the film, the coverslip is washed to remove free particles, and 40 nmbiotin labeled (fluorescent) particles are then used to specificallyrecognize the avidin pattern. 16.40 nm avidin coated (non-fluorescent)particles and 40 nm biotin coated (fluorescent) particles (Molecularprobes) were stored in 50 mM sodium phosphate buffer, 50 mM NaCl, 7.4pH, 0.02% Tween (polyoxyethylenesorbitan, including fatty acid estersthereof), 2 mM sodium azide. The sample volume is 10 μl corresponding to10¹² particles.

The avidin-biotin pair was chosen as a model of receptor-ligandbiomolecular system. This process is schematically shown in FIGS. 3A-3C.FIG. 3D shows a typical result, while FIG. 3E shows the effect ofvarying the spot size and correspondingly varying the line width. Thelimitation on the separation between lines is defined by the spot size,and is on the order of a micron. Subsequent writing with a micron scaleline separation does not alter the bio-specificity of the writtenlithographic patterns. As a control, after inscription and recognition,sample temperature was increased up to 65° C. and the dissociation ofthe biotin-avidin binding was observed, probably due to denaturation ofavidin.

The second example set forth herein deals with the problem ofrecognition of a given sequence of single stranded DNA by hybridizationto the complementary strand. Two different nucleic acid molecules, onemolecule comprising a DNA sequence of GTATCACGAGGCCCT (SEQ ID NO:1) andthe other molecule comprising a DNA sequence of GACAGCTTATCATCG (SEQ IDNO:2) were used to demonstrate the specificity of recognition. Particlescovered with the nucleic acid molecule comprising a DNA sequence of SEQID NO:1 were initially deposited on even lines of an array of parallellines using the apparatus and method of the present invention. Theparticle-oligo construct involved 10 μl of a stock of biotinylatedoligomers (0.05 mg/ml in TE buffer) added to 10 μl of avidin coatedparticles (10¹² particles), which was then incubated at room temperaturefor 10 minutes. The construct is identical for SEQ ID NO:1 and SEQ IDNO:2.

After washing the sample, particles coated with the nucleic acidmolecule comprising a DNA of SEQ ID NO:2 were deposited on the odd lines(FIG. 4A). To recognize the patterned single stranded DNA, DNA moleculescomplementary to SEQ ID NOS:1 and 2 were used. The complementarysequences were tagged by two different fluorophores at the 3' end, FAMfor the DNA molecule complementary to SEQ ID NO:1, and Rhodamine for theDNA molecule complementary to SEQ ID NO:2. This is depicted in FIG. 4B.Adding equal amounts of the two complementary sequences to the samplewell leads to specific recognition of the alternating inscribed DNApatterns. The resulting fluorescence microscopy picture is shown in FIG.4C. The images are simultaneously acquired, in grayscale, using twodifferent excitation filters to detect the hybridization of the twosequences.

Detection involved introducing nucleic acid molecules having DNAsequences complementary of both oligonucleotides (0.05 mg/ml in TEbuffer 20 μl volume) into the sample cell. The hybridization reaction iscarried out for approximately one hour at room temperature. Afterrinsing, detection is done using standard fluorescence microscopy andimage intensifier (Hamamatsu). FAM is excited at 480 nm (fluoresceinfilter) and Rhodamine at 530 nm (Rhodamine filter).

The grayscale images are then independently colored to represent the twofluorophores and then added to show the sequence specificity of theetched patterns (FIG. 4C). Non-specific interactions lead to much weakercontrast. Differentiation is easily detected, even for 5 μm separationbetween two neighboring oligomer lines.

EXAMPLE II Biochip Microfabrication: grafting of particles onto asemiconductor substrate

DNA sequences or proteins serve as templates for information retrievalusing specific recognition, i.e. hybridization for DNA and bindingaffinity for proteins. The possibility to assemble and recognize largenumber of DNA or protein molecules, in an addressable array on a solidsubstrate has wide ranging applications in biology (2). This isparticularly important in high throughput screening for mutations, generearrangements, gene expression, viral integration or diversity inprotein sequences. Recent progress has been made possible by applyingchemical techniques to synthesize DNA sequences on a substrate(1,12-16). Presented here is a novel method to microfabricate DNA/proteinchips for a variety of applications. The present invention also allowsthe development of ultrasmall biochips and biosensors. Results andDiscussion

The present invention is based upon Applicants' discovery that by usingan infrared optical tweezer (17), one can select a Brownian colloidalinsoluble particle in a colloidal dispersion and use the same tweezer toselectively graft the particle into a silicon substrate (such as asilicon cantilever used in atomic force microscopy(18)). Particlegrafting is done using the tweezer to heat the cantilever tip byinfrared absorption. As it is heated, the polystyrene latex particleadheres to the cantilever by polymer wetting (FIG. 5). The particlewetting properties are not altered by the presence of a layer ofadsorbed DNA or proteins. The method is general and does not depend onthe substrate used as long as it absorbs infrared radiation. The utilityof the present invention is demonstrated herein by grafting twoparticles with different single-stranded (ss) oligonucleotides to asubstrate. The molecules on the particles are then exposed tofluorescently labeled complementary sequences for detection. Standardforce microscope silicon cantilevers are used as semiconductorsubstrates (a silicon tip or a silicon nitride tip on one side andsub-micron thickness gold coating on the other side). A thin filmdeposited on the substrate, such as a thin gold coating, enhances theinfrared absorption, both at the gold silicon interface and byincreasing reflection. Typical incident laser diode power used forgrafting is ˜70 mW. The grafting time depends on the volume of siliconto be heated. To graft a 3-μm polystyrene particle to the tip of thesilicon cantilever takes about ˜0.2 seconds (tip characteristic length 3μm, curvature at the tip about 20 nanometers).

To demonstrate the feasibility of the method particles, two differentknown DNA sequences were grafted onto a silicon cantilever. The DNAmolecules coating the particles were then exposed to DNA moleculescomplementary thereto, and tagged with a fluorophore at the 3' end (FIG.6).

The detailed procedure goes as follows: Biotin-modified ss DNA sequencesare first biochemically attached to streptavidin-coated polystyreneparticles. Using a small flow cell, a first colloidal dispersion withparticles covered with a DNA molecule having a known sequence aretransferred to a sample chamber. An optical tweezer is used to selectone particle and then graft it to a pre-mounted cantilever in thechamber. In the next step, the chamber is washed with phosphate bufferedsaline (PBS) and a second colloidal dispersion with a set of particlescoated with a second DNA molecule having a known sequence is transferredto the sample chamber for physical grafting. The grafted sequences arethen detected by hybridization of ss, fluorescent DNA, using standardbuffer conditions (see FIG. 6). During the physical grafting, there isminimal rupture of the functionality of the biotin-streptavidin links orthe chemical bonds in DNA. This observation has been verified even atthe level of a single double-stranded DNA polymer attachment (11). Thetime required to assemble an array of n particles, scales as n. It isestimated that with automated approaches, a typical time to graft 10⁴particles in a regular array would be about 1 hour.

In summary the present invention readily permits grafting arrays ofgenomic DNA and proteins for real-time process monitoring based onDNA-DNA, DNA-protein or receptor-ligand interactions. By using anoptical tweezer as a noninvasive tool, a particle coated with amolecule, such as a biomolecule, can be selected and grafted ontospatially localized positions of a semiconductor substrate. Thenoninvasive optical method, in addition to biochip fabrication, hasapplications in grafting arrays of specific biomolecules withinmicrofluidic chambers that may enable separation methods for moleculesas well as cells.

Experimental Protocol

Grafting particles: The optical tweezer is constructed using a 150 mW,830 nm near-infrared laser diode (SDL) and a 100 X, 1.3 N.A microscopeobjective (Zeiss). The wavelength of the infrared laser used correspondsto an energy of 1.48 eV. With silicon substrates (FIG. 6), about 10% ofthe incident power (˜70 mW) is absorbed (Ia=Io (1-e.sup.α1)≦0.1, here Iais the absorbed power, Io is the incident power on a 5-μm² surface areaof silicon cantilever, α=780 cm⁻¹ is the absorption coefficient at 830nm and t=0.8 μm, the thickness of the silicon cantilever) (19). In thiscase the increase in temperature of the flat silicon substrate insolution, for one second irradiation, is about 40° C. The sample-chamberis formed by a #1 coverglass with a polyvinyline o-ring glued to it byparaffin. The silicon substrate used is a standard silicon / siliconnitride cantilever (Park Scientific). It is mounted on a precision XYZstage for alignment of the substrate with the laser tweezer axis. Byusing a small flow tube, particles covered with known sequence andsuspended in 0.1 M PBS, are transferred to the sample chamber. A singleparticle is trapped using the laser tweezer and the aligned cantilevertip is moved to the trap. Typical grafting time is ˜0.2 seconds. Thesample chamber is rinsed in 0.1 M PBS and a second set of particles isintroduced for grafting. The grafting and washing procedures are donemanually.

Particle Preparation

3-μm amino modified polystyrene particles (Polysciences) are firstcovered with streptavidin by covalent attachment using glutaraldehydelinker. Biotin modified oligomers (New England Biolabs) are mixed withthe particles (˜10⁶ oligomers/bead). Oligomers can then be attachedusing the biotin-streptavidin linker. Particles are suspended in PBSafter centrifugation to remove free oligomers in solution and aredirectly used for grafting.

Detection

Complementary sequences are labeled with fluorescent markers forhybridization detection. The complement to a DNA molecule comprising aDNA sequence of GTATCACGAGGCCCT (SEQ ID NO:1) is labeled with FAM and acomplement to a DNA molecule comprising a DNA sequence ofGACAGCTTATCATCG (SEQ ID NO:2) is labeled with rhodamine, wherein bothcomplements are labeled at their 3' end (New England Biolabs). After theparticles are grafted, complementary strands of DNA (10 μg/ml in 0.1 MPBS) are transferred to the sample chamber for hybridization. Thereaction is carried out at room temperature for more than 1 hour and thesample chamber is rinsed with PBS to remove any background fluorescencesignal. The hybridization is detected with a standard fluorescencemicroscope arrangement with an intensifier and camera (Hamamatsu). Thegrafted beads are excited at 480 nm using a fluorescein filter tovisualize FAM and then with 530 nm using a rhodamine filter to visualizerhodamine. Specific recognition of the two sequences on the graftedparticles results in fluorescence. The data are recorded in grey scaleon a video tape and artificially colored to show the contrastdifference. Non-specific interaction results in much weaker contrast.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Furthermore, it is to be understood that the invention is not limited tothe illustrations described and shown herein, which are deemed to bemerely illustrative of the best modes of carrying out the invention, andwhich are susceptible of modification of form, size, arrangement ofparts and details of operation. The invention rather is intended toencompass all such modifications which are within its spirit and scopeas defined by the appended claims.

Various publications are cited herein, the disclosures of which arcincorporated by reference in their entireties.

REFERENCES

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What is claimed is:
 1. Apparatus for immobilizing a molecule onto asubstrate, comprising:a) means for directing a beam of electromagneticradiation at a substrate, wherein said substrate comprises a firstsurface and a second surface, and a film of a material deposited ontosaid second surface, wherein said film absorbs electromagnetic radiationand is a poor conductor of heat; and b) a chamber for holding acolloidal dispersion comprising insoluble particles coated with saidmolecule, such that said colloidal dispersion is in contact with saidfilm; such that said beam can impinge said first surface, traverse saidsubstrate, impinge an area of said film and ablate and melt said film atsaid area so that a gas bubble is formed in said colloidal dispersionabove said area and disappears upon displacement of said beam, and saidparticles can adhere to said film at said area so that said molecule isimmobilized onto said substrate.
 2. The apparatus of claim 1, whereinsaid substrate comprises glass or silica.
 3. The apparatus of claim 1,wherein said film has a thickness of approximately 50 Å.
 4. Theapparatus of claim 1, wherein said film comprises a material thatabsorbs electromagnetic radiation, is a poor conductor of heat, and hasa low melting temperature.
 5. The apparatus of claim 4, wherein saidmaterial comprises gold or aluminum.
 6. The apparatus of claim 1,wherein said colloidal dispersion comprises an aqueous solution.
 7. Theapparatus of claim 6, wherein said aqueous solution comprises 50 mM of aphosphate buffered saline PBS), 50 mM NaCl, 0.2% polyoxyethylenesorbitan, 2 mM NaN₃, pH=7.4, or 0.1 M PBS.
 8. The apparatus of claim 1, whereinsaid insoluble particles have a size smaller than said area.
 9. Theapparatus of claim 8, wherein insoluble particles have a size ofapproximately 2 nm to approximately 100 nm.
 10. The apparatus of claim1, wherein said particles comprise polystyrene, gold, or glass.
 11. Theapparatus of claim 1, wherein said particles comprise polystyrene beadshaving a size of about approximately 40 nm.
 12. The apparatus of claim1, wherein said colloidal dispersion comprises a concentration ofparticles of approximately 10¹¹ particles/μl.
 13. The apparatus of claim1, wherein said beam has a wavelength in the near infrared region of theelectromagnetic spectrum.
 14. The apparatus of claim 13, wherein saidbeam of comprises a laser beam comprising a wavelength of 830 nm or 1064nm.
 15. The apparatus of claim 14, wherein said laser beam comprises awavelength of 830 nm, an incident power of approximately 35 mW, amaximum power of approximately 150 mW, a circular cross sectional areawith a diameter of approximately 1 μm, and impinges said area forapproximately 0.2 seconds.
 16. The apparatus of claim 1, wherein saiddirecting means comprises a microscopic objective lens immersed in oil,wherein said lens has a magnification of 100X, a 1.3 N.A., and a focalpoint on said film.
 17. The apparatus of claim 1, further comprising ameans for steering said beam to permit movement of impingement of saidbeam with said film.
 18. The apparatus of claim 17, wherein saidsteering means comprises two telescopic lenses with a focal length of100 mm positioned such that said beam propagates through said telescopiclenses prior to propagating through said directing means.
 19. Theapparatus of claim 17, wherein movement of impingement of said beam withsaid film comprises a velocity of approximately 5 μm/second toapproximately 50 μm/second.
 20. The apparatus of claim 1, furthercomprising a means of generating said beam.
 21. The apparatus of claim20, wherein said generating means comprises a near infrared diode laser.22. The apparatus of claim 1, wherein said molecule is a biomolecule.23. The apparatus of claim 22, wherein said biomolecule comprises anisolated nucleic acid molecule or fragments thereof, an isolated proteinor fragments thereof, a lipid, or a carbohydrate.
 24. The apparatus ofclaim 23, wherein said isolated nucleic acid molecule comprises DNA,RNA, or a combination thereof.
 25. The apparatus of claim 23, whereinsaid isolated protein comprises an antibody, a receptor, a DNA bindingprotein, a protein having a protein binding domain, a cytokine, alymphokine, or a hormone.
 26. Apparatus for immobilizing a molecule ontoa substrate, comprising:a) a chamber for holding a colloidal dispersionof insoluble particles coated with said molecule; b) means forsupporting said substrate in said chamber; and b) means for selecting aparticle of said colloidal dispersion and grafting said particle ontosaid substrate, such that said molecule is immobilized onto saidsubstrate.
 27. The apparatus of claim 26, wherein said substratecomprises a cantilever, and said particle is grafted to said cantilever.28. The apparatus of claim 26, wherein said substrate comprises siliconor silicon nitride.
 29. The apparatus of claim 27, wherein saidsubstrate is coated with a film that absorbs electromagnetic radiation,is a poor conductor of heat, and has a low melting temperature.
 30. Theapparatus of claim 29, wherein said film comprises gold or aluminum. 31.The apparatus of claim 29, wherein said film has a thickness ofapproximately 50 Å.
 32. The apparatus of claim 27, wherein saidcolloidal dispersion comprises an aqueous solution.
 33. The apparatus ofclaim 32, wherein said aqueous solution comprises 50 mM of a phosphatebuffered saline (PBS), 50 mM NaCl, 0.02% polyoxyethylenesorbitan, 2 mMNaN₃,pH=7.4 or 0.1 M PBS.
 34. The apparatus of claim 27, wherein saidparticles comprise polystyrene, gold, or glass.
 35. The apparatus ofclaim 33, wherein said particles comprise polystyrene beads having asize of about approximately 3 μm.
 36. The apparatus of claim 27, whereinsaid selecting and grafting means comprises an optical tweezer.
 37. Theapparatus of claim 36, wherein said optical tweezer comprises a laserbeam having a wavelength in the near infrared region of theelectromagnetic spectrum, and a microscopic objective lens, such thatsaid beam passes through said lens, and enters said chamber.
 38. Theapparatus of claim 37, wherein said microscopic objective lens hasproperties of 100 X, and 1.3 numerical aperture.
 39. The apparatus ofclaim 37, wherein said laser beam comprises a wavelength of 830 nm. 40.The apparatus of claim 27, wherein said chamber is formed by a #1 coverglass with a polyvinyline o-ring glued onto said cover glass withparaffin.
 41. The apparatus of claim 27, wherein said supporting meanscomprises an XYZ stage located adjacent to said chamber, wherein saidstage permits movement of said substrate towards said selected particle.